Anti-ultraviolet radiation beverage and preparation method thereof

Abstract

The invention relates to an anti-ultraviolet radiation beverage and a preparation method thereof.The anti-ultraviolet radiation beverage comprises high-purity metallothionein prepared from sturgeon livers through the steps of tissue breaking, step-by-step composite enzymolysis, multistage centrifugation, filtration and the like, and the preparation method specifically comprises the steps that after fresh sturgeon livers are broken into homogenate, a compound enzyme compounded by neutral protease and flavourzyme is added, carrying out step-by-step enzymolysis at the constant temperature of 50-55 DEG C for 3-4 hours, carrying out high-speed centrifugal separation on supernate, and filtering through a 0.45 mu m filter membrane; and carrying out heating inactivation and ice bath cooling on the filtrate, and then carrying out centrifugal freeze-drying to obtain the metallothionein. According to the technology, the extraction efficiency and activity of metallothionein are remarkably improved by optimizing enzymolysis conditions and a purification process, and the obtained beverage can effectively remove free radicals induced by ultraviolet rays and enhance the ultraviolet radiation resistance of skin, is suitable for the fields of outdoor activities, daily sunscreen, skin health protection and the like, and has the dual advantages of nutrition and functionality.

Description

Technical Field

[0001] The present invention relates to the technical field of functional beverage preparation, and more particularly to an anti-ultraviolet radiation beverage and a preparation method thereof. Background Art

[0002] Ultraviolet radiation damage to human skin has become a significant issue in the field of environmental health. Long-term exposure to UV rays can lead to photoaging, DNA damage, and inflammatory responses. Traditional UV protection products rely primarily on chemical sunscreens (such as oxybenzones) or plant-derived antioxidants (such as polyphenols). However, chemical sunscreens pose potential allergenic risks and may disrupt the endocrine system. Plant extracts are limited by poor ingredient stability and low bioavailability, requiring high concentrations to achieve their protective effects. However, high doses can introduce a bitter taste or react with other ingredients, affecting product palatability. Therefore, there is an urgent need to develop safe, effective, and well-compatible natural anti-UV active ingredients.

[0003] Metallothioneins (MTs) are a class of small, metal-binding proteins rich in cysteine residues. They possess biological functions such as free radical scavenging, heavy metal chelation, and UV protection. Their thiol groups can directly neutralize UV-induced reactive oxygen species (ROS), mitigating oxidative damage. However, the large-scale production of MTs faces significant technical bottlenecks. Firstly, the limited content of MTs in mammalian liver (the traditional source of extraction) raises ethical concerns. Secondly, while aquaculture byproducts (such as fish liver) are rich in MTs, their high-purity, high-activity production is difficult due to inadequate raw material pretreatment techniques, low extraction efficiency, and protein volatility. The main challenges in extracting metallothioneins from fish livers include: 1. Sturgeon liver tissue is dense, and conventional mechanical disruption can lead to incomplete disruption of the cell structure and incomplete release of metallothioneins. Excessive disruption can cause protein denaturation due to frictional heat generation, while improper intermittent cooling can lead to autolysis of the raw material before enzymatic hydrolysis, reducing the yield of the target protein. 2. The extraction of metallothioneins relies on the degradation of the liver tissue matrix by specific proteases. However, single proteases (such as neutral proteases) have limited sites for action, making it difficult to fully release target proteins bound to organelles. Furthermore, pH and temperature fluctuations during the enzymatic hydrolysis process can significantly affect enzyme activity. Conventional one-time enzyme addition methods can easily lead to excessive enzyme consumption in the initial stage, while insufficient enzyme activity in the later stages can leave undegraded protein impurities in the final product, complicating subsequent purification. 3. Metallothioneins are sensitive to heat and oxidation, making conventional centrifugation and filtration processes difficult to remove large molecular impurities while maintaining their structural stability. For example, inaccurate temperature control during the high-temperature enzyme inactivation step can lead to protein aggregation and precipitation. In conventional freeze-drying processes, the lack of protective agents can easily lead to protein inactivation due to ice crystal formation. Furthermore, residual fat particles and polysaccharides in the final product will compete with metallothioneins for metal ion binding, reducing their functional activity. In liquid beverages, metallothioneins are susceptible to changes in pH, ionic strength, and coexisting ingredients (such as fruit acids and sugars), causing conformational changes or precipitation, leading to decreased bioavailability. Existing technologies often use encapsulation or microencapsulation to improve stability, but these additional processing steps increase costs and may introduce exogenous excipients, contradicting the product's positioning as natural and healthy. The root of the above problems lies in the inherent contradiction between the physical and chemical properties of metallothioneins and the complexity of the tissues from which they are derived: on the one hand, the target protein needs to be released by efficiently destroying the cell structure, and on the other hand, denaturation caused by oxidation, heat, and mechanical shearing needs to be avoided throughout the extraction process. Existing processes often compromise one aspect while focusing on the other. For example, extending the enzymatic hydrolysis time to increase the extraction rate in turn exacerbates the degradation of metallothioneins by proteases; or omitting the precise temperature control step to shorten the process, resulting in loss of activity. In addition, optimizing the stability of metallothioneins in beverage formulations requires balancing their interactions with ingredients such as fruit pulp and acidulants. This places higher demands on the purity of the extraction process and the conformation of the product, further increasing the difficulty of technical integration. Therefore, developing a method for preparing metallothionein that can take into account extraction efficiency, product activity and beverage compatibility has become a technical difficulty that urgently needs to be overcome in this field. Summary of the Invention

[0004] The object of the present invention is to provide an anti-ultraviolet radiation beverage and a preparation method thereof, so as to at least solve the above problems.

[0005] In order to achieve the purpose and other advantages of the present invention, an anti-ultraviolet radiation beverage is provided, comprising: metallothionein at a concentration of 95-105 mg / mL, wherein the preparation method of the metallothionein comprises the following steps: Wash the fresh sturgeon liver, cut it into pieces, and crush it with a tissue crusher to form a tissue homogenate; The tissue homogenate is heated to 50-55° C., the pH value of the tissue homogenate is adjusted to 6.5-7.0, and a complex enzyme is added in two portions, with the first addition amount being 60%-70% of the total weight of the complex enzyme. After 1-1.5 hours of enzymatic hydrolysis, the remaining complex enzyme is added, wherein the complex enzyme comprises a neutral protease and a flavor protease in a mass ratio of 1:1, and the addition amount of the complex enzyme is 0.5%-1.0% of the weight of the sturgeon liver. After adding the complex enzyme, the enzymatic hydrolysis is carried out by stirring at a speed of 150-200 rpm for a total enzymatic hydrolysis time of 3-4 hours to obtain an enzymatic hydrolyzate. The enzymatic hydrolyzate was centrifuged at 8000-10000 rpm for 15-20 minutes at 4°C to obtain a supernatant; The supernatant was preliminarily filtered through gauze, and then filtered through a 0.45 μm microporous filter membrane to obtain a filtrate; The filtrate was heated in a water bath at 80-85°C for 10-15 minutes with continuous stirring, and then cooled to room temperature in an ice bath; The cooled filtrate is centrifuged at 4° C. and 10,000-12,000 rpm for 10-15 minutes, and the supernatant is collected and freeze-dried to obtain the metallothionein.

[0006] Preferably, the fresh sturgeon liver is cleaned and cut into 0.5-1 cm 3 The liver was pounded into cubes in two steps using a tissue pounder. The first pounding time was 1.5-2 minutes in an intermittent pulse mode of 5 seconds on / 3 seconds off, with a pounder speed of 3000-3500 rpm. The second pounding time was 1.5-3 minutes in a continuous rotation mode, with a pounder speed of 4000-4500 rpm. Before each pounding, the sturgeon liver blocks or tissue homogenate were transferred to a mixture of crushed ice and water and allowed to stand until the core temperature dropped to 4-6°C.

[0007] Preferably, before heating the tissue homogenate, the tissue homogenate is also subjected to ultrasonic pretreatment, and the conditions of the ultrasonic pretreatment are: frequency 20-25kHz, power density 50-80W / L, and treatment time 8-12 minutes; wherein, during the ultrasonic pretreatment process, the temperature of the tissue homogenate is maintained at 4-6°C.

[0008] Preferably, when adjusting the pH value of the tissue homogenate, the pH is first pre-adjusted to 6.0±0.2 with 0.1M citric acid-disodium hydrogen phosphate buffer, and then 0.1M sodium bicarbonate solution is added dropwise at a rate of 0.5mL / min through a microinjection pump to a target pH value of 6.8±0.1; and after the first addition of the complex enzyme, 0.02%-0.05% of the mass of the tissue homogenate is added to the enzymatic hydrolysis solution every 45 minutes. The mercaptoethanol protective agent is prepared by compounding L-cysteine and reduced glutathione in a molar ratio of 3:1.

[0009] Preferably, the complex enzyme needs to be pre-activated before addition: the neutral protease and the flavor protease are activated separately at 40° C. and pH 7.0 for 15 minutes, and then mixed in a ratio of 1:1; the stirring enzymatic hydrolysis is divided into two stages: stirring at 150-170 rpm after the first addition of the complex enzyme, and stirring at 180-200 rpm after the second addition of the complex enzyme; during the total enzymatic hydrolysis time, the temperature is maintained at 50-52° C. for the first 2 hours and then raised to 54-55° C. for the next 1-2 hours.

[0010] Preferably, before the cooled filtrate is centrifuged at 10,000-12,000 rpm at 4°C for 10-15 minutes, the filtrate is first centrifuged at 8,000-10,000 rpm at 4°C for 5-8 minutes, the precipitate is discarded, and the resulting supernatant is centrifuged at 10,000-12,000 rpm. After centrifugation, the supernatant is filtered through a 0.22 μm sterile filter membrane for terminal sterilization to obtain a final filtrate, and a lyophilization protectant is added to the final filtrate before freeze-drying, wherein the lyophilization protectant is composed of trehalose and mannitol in a mass ratio of 1:2, and the amount added is 5%-8% of the protein mass in the final filtrate.

[0011] Preferably, the anti-ultraviolet radiation beverage further comprises: mixed fruit puree, wherein the mixed fruit puree comprises, by volume percentage, 30-40% blueberry puree, 25-35% mulberry puree, and the remainder black wolfberry puree.

[0012] The present invention also provides a method for preparing an anti-ultraviolet radiation beverage, which is characterized by comprising the following steps: Add blueberry puree, mulberry puree, and black wolfberry puree according to the formulated amount into a mixing tank and stir at 200-300 rpm for 15-20 minutes to form a mixed fruit puree; add metallothionein powder to the mixed fruit puree to a final concentration of 95-105 mg / mL of metallothionein and continue stirring for 10-15 minutes; then add 3-5% sucrose and 0.1-0.3% citric acid of the total weight of the mixed fruit puree in sequence, and stir at 150-200 rpm at 50-60° C. for 20-30 minutes; The prepared mixed liquid is transported to a high-pressure homogenizer through a pipeline and subjected to two-stage homogenization treatment at a pressure of 20-30 MPa and a temperature of 40-50°C. The first-stage homogenization pressure is 25-28 MPa and the gap of the homogenization cavity is 0.1 mm. The second-stage homogenization pressure is 18-22 MPa and the gap of the homogenization cavity is 0.25 mm. The interval between the two-stage homogenization treatments does not exceed 5 minutes. The homogenized mixed liquid is preheated to 75-80°C through a plate heat exchanger, transported to a high-temperature instantaneous sterilization device through a centrifugal pump, maintained at 120-130°C for 3-5 seconds, and immediately cooled to 25-30°C within 45 seconds through a spiral thin-layer heat exchanger; the cooled mixed liquid is filled into glass bottles sterilized with 121°C steam for 20 minutes, and the temperature of the mixed liquid is maintained at 25-30°C during filling. Immediately after filling, the headspace air is replaced with nitrogen until the residual oxygen content is ≤0.8%, and the bottles are sealed with screw caps. Preferably, the method for preparing the anti-ultraviolet radiation beverage further includes, in the step of adding sucrose and citric acid, simultaneously adding 0.05-0.1% of vitamin C, 0.02-0.05% of vitamin E and 0.8-1.2% of β-cyclodextrin based on the total mass of the mixed fruit pulp, wherein the vitamin C and vitamin E are pre-dry-mixed in a mass ratio of 2:1 and then added, and stirring is maintained at 55-60°C for 25 minutes.

[0013] Preferably, the β-cyclodextrin is pretreated before being added, specifically comprising the following steps: mixing β-cyclodextrin with 40-45°C water in a mass-to-volume ratio of 1:10, stirring and swelling in a water bath at 100-150 rpm, controlling the water temperature to rise from the initial temperature to 40°C at a rate of 1°C per minute, and maintaining at this temperature for 15 minutes; then heating to 60°C at a rate of 2°C per minute, adjusting the stirring speed to 80-100 rpm, and maintaining constant temperature stirring for 10 minutes; stopping heating when the solution transmittance reaches more than 90%, and naturally cooling to 45-50°C under continuous stirring to form a transparent inclusion solution, and immediately transferring it into a mixing tank.

[0014] The present invention has at least the following beneficial effects: First, the release efficiency of metallothionein from sturgeon liver was optimized by adding complex enzymes in stages and combining temperature and pH control. Neutral proteases and flavor proteases work synergistically, with the former cutting the internal peptide bonds of the protein and the latter degrading the hydrophobic terminal peptide chains, thereby more thoroughly hydrolyzing the liver tissue matrix and reducing the amount of undegraded impurities remaining. The strategy of adding enzymes in stages can avoid self-consumption caused by excessive enzymes, maintain the activity in the later stages of enzymatic hydrolysis, and improve the yield of the target protein. In addition, the gradient centrifugation and heat treatment steps after enzymatic hydrolysis can effectively separate impurities with poor thermal stability, and fix the conformation of metallothionein by rapid cooling, reducing oxidative damage. The final freeze-drying process combined with low-temperature centrifugation can retain the activity of metallothionein to the greatest extent, providing high-purity raw materials for subsequent beverage formulations.

[0015] Second, through staged mechanical crushing and temperature control, refined processing of sturgeon liver tissue was achieved. The first pulse crushing combined with intermittent cooling can avoid local overheating caused by continuous high-speed shearing and reduce the risk of protein thermal denaturation; the second continuous high-speed crushing further refines tissue particles and improves homogenate uniformity. The combination of block cutting and low-temperature stasis can inhibit the autolysis of endogenous enzymes while maintaining the integrity of cell structure, ensuring the targeted release of metallothioneins during subsequent enzymatic hydrolysis. This method improves crushing efficiency while reducing the damage to the target protein caused by mechanical energy input, laying the foundation for efficient extraction.

[0016] Third, ultrasonic pretreatment enhances cell membrane permeability, promoting the contact efficiency between the enzymatic substrate and the complex enzyme. The cavitation effect of ultrasound can generate microjets and shear forces, physically destroying the cell wall and organelle membrane structure without significantly increasing the temperature, releasing more bound metallothioneins. Simultaneously, the low temperature environment inhibits oxidation reactions that may be triggered during the ultrasound process, preventing the loss of sulfhydryl groups. This step shortens the time required for subsequent enzymatic hydrolysis and reduces the amount of enzyme required, thereby improving extraction efficiency and reducing process costs.

[0017] Fourth, the pH value is precisely controlled using a buffer and a microinjection pump to ensure the enzymatic hydrolysis system is within the optimal activity range, avoiding enzyme inactivation or protein denaturation caused by drastic pH fluctuations. A phased adjustment strategy reduces the problem of localized excess concentrations of acid and base reagents and maintains reaction uniformity. Regular addition of mercaptoethanol protective agents (L-cysteine and reduced glutathione) dynamically scavenges free radicals, protecting the active sulfhydryl groups of metallothioneins from oxidative damage. The synergistic effect of the combined protective agents is more effective than that of each individual component, further stabilizing protein conformation and enhancing the antioxidant capacity of the final product.

[0018] Fifth, the catalytic efficiency of the enzyme complex was optimized through enzyme preactivation and a phased stirring strategy. After activation, the neutral protease and flavor protease rapidly enter a highly active state, shortening the lag phase of the enzymatic hydrolysis reaction. Adjustment of the two-stage stirring speeds matched the requirements of the enzymatic hydrolysis process: initial low-speed stirring prevented excessive shear damage to the released metallothioneins, while accelerated stirring in the later stages promoted further hydrolysis of undegraded tissue residues. The temperature gradient design (50-52°C in the early stages, 54-55°C in the later stages) balanced the optimal range of enzyme activity with the thermal stability of the target protein, minimizing the co-dissolution of contaminants.

[0019] Sixth, the purity of metallothionein was significantly improved by combining multi-stage centrifugation with terminal filtration. After initial centrifugation to remove large impurities, secondary high-speed centrifugation separated small lipids and polysaccharides, reducing interfering substances that competitively bind to metal ions in the final product. Trehalose and mannitol were added as lyoprotectants, binding to the protein surface through hydrogen bonds, reducing mechanical damage to the protein structure caused by ice crystal formation and maintaining the rehydration properties of the protein after lyophilization. Terminal sterile filtration avoided the negative effects of high-temperature sterilization on protein activity, ensuring the microbial safety of the final product.

[0020] Seventh, through the blending of mixed fruit purees, a balance of functionality and palatability is achieved. Blueberry, mulberry, and black wolfberry purees are rich in anthocyanins and polyphenols, which synergize with the antioxidant mechanism of metallothioneins to enhance overall UV protection. The natural acidity of the purees adjusts the pH of the beverage to the stable range of metallothioneins (near neutral), reducing the need for the addition of separate acidifiers. Furthermore, the viscous texture of the purees partially inhibits the Brownian motion of protein molecules, reducing the risk of aggregation and precipitation, while masking the potential metallic odor of metallothioneins and enhancing consumer acceptance.

[0021] Eighth, a two-stage high-pressure homogenization and instantaneous sterilization process ensures the physical stability and biosafety of the beverage system. The first high-pressure homogenization stage breaks down pulp fibers and protein aggregates, while the second lower-pressure homogenization stage refines the particle distribution, creating a uniform dispersion and delaying sedimentation and stratification. High-temperature instantaneous sterilization inactivates microorganisms while shortening the exposure time of heat-sensitive ingredients (such as pulp pigments and metallothioneins), minimizing activity loss. Nitrogen displacement of headspace oxygen inhibits oxidation, extending shelf life, and eliminating the need for traditional preservatives, in line with the clean label trend.

[0022] Ninth, the addition of vitamins C, E, and β-cyclodextrin creates a multi-factor stabilization mechanism. The synergistic antioxidant effects of vitamins C and E protect metallothioneins and fruit pulp components from oxidative degradation during processing and storage. β-cyclodextrin encapsulates the hydrophobic regions of metallothioneins within its hydrophobic cavity, reducing direct contact with fruit acids or metal ions and inhibiting precipitation caused by conformational changes. Furthermore, β-cyclodextrin can mask some of the bitterness and improve the flavor of the beverage. The dry-mix addition method prevents premature oxidation of the liquid vitamins, ensuring the effectiveness of the functional ingredients.

[0023] Tenth, β-cyclodextrin is pretreated through a gradient temperature ramp to optimize its inclusion capacity and solubility. The low-temperature swelling stage fully hydrates the cyclodextrin molecules, opening up their rigid ring structure. The subsequent stepwise temperature ramp promotes dynamic binding with the target molecule, forming a stable inclusion complex. This process avoids the cyclodextrin's coking or agglomeration that can occur with direct high-temperature treatment, ensuring high transparency and stability of the inclusion solution. The pretreated β-cyclodextrin disperses rapidly in the beverage system, reducing stirring energy consumption during blending and improving inclusion efficiency, thereby more effectively protecting the activity of metallothioneins.

[0024] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to the embodiments, comparative examples and experimental examples so that those skilled in the art can implement the invention with reference to the description.

[0026] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0027] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0028] In one embodiment of the present invention, during the raw material pretreatment step, the sturgeon liver cutting size can be set to 0.8-1.2 cm 3The cubes are crushed in two steps: the first using intermittent pulse mode (5 seconds on / 3 seconds off) at 3200 rpm for 1.8 minutes; the second using continuous mode at 4200 rpm for 2.5 minutes. A high-speed tissue crusher equipped with 304 stainless steel blades and a variable-frequency motor is recommended. The ratio of blade diameter to tank inner diameter is 1:4. Sturgeon livers are sourced from farmed Siberian sturgeons and processed within 2 hours of slaughter. The crusher is located in the pretreatment area, connected to the cutting station by a conveyor belt and connected downstream to a constant-temperature water bath. The cut liver chunks enter the crusher through a hopper. The blades rotate intermittently in pulse mode to prevent overheating. The second continuous crushing step refines the tissue particles, and the homogenate is then discharged into a sandwich reactor through the discharge port. During the stepwise enzymatic hydrolysis process, the initial enzyme addition amount is 65% of the total enzyme mass. The remaining 35% is added after 1.2 hours of hydrolysis. The enzyme mass is premixed with a neutral protease (enzyme activity ≥ 500,000 U / g) and a flavor protease (enzyme activity ≥ 200,000 U / g) in a 1:1 mass ratio. A double-layer stirred enzymatic hydrolysis tank can be used, with an anchor-type + pitched-blade turbine impeller combination, operating at 160 rpm in the first stage and 190 rpm in the second stage. The enzymatic hydrolysis tank is installed in a constant temperature chamber, with a temperature probe located on the tank side and a pH electrode inserted 10 cm below the liquid surface. A PLC program controls the enzyme addition time and stirring gradient. The temperature is set at 51 ± 0.3°C for the first 2 hours and then increased to 54.5 ± 0.3°C over the next 1.5 hours. For experimental validation, three batches of homogenate samples were collected and tested for metallothionein release at different enzyme addition ratios (60%, 65%, and 70%). Protein concentration was determined using the BCA assay. During the isolation and purification step, the enzymatic hydrolysate is first centrifuged at 9000 rpm for 18 minutes at 4°C. The supernatant is then filtered through a 0.45 μm microporous membrane and heated in a water bath at 82 ± 1°C for 12 minutes, followed by an ice bath cooling rate of ≥5°C / minute. A high-speed refrigerated centrifuge with a 6 × 500 mL rotor and a membrane made of mixed cellulose esters can be used. Trehalose and mannitol (mass ratio 1:2) are added before lyophilization. The lyophilization process involves a pre-freezing phase at -40°C for 4 hours, followed by a gradual temperature increase to 20°C during the sublimation phase. The centrifuge is located in a clean area adjacent to the enzymatic hydrolysis tank, with the filtration device connected in series to the centrifuge outlet. The heat-treated filtrate is rapidly cooled in an ice bath and centrifuged a second time at 11,000 rpm for 12 minutes. The supernatant is sterilized through a 0.22 μm sterile filter and then lyophilized. This process reduces heat-sensitive impurities through fractionation and low-temperature treatment, maintaining the sulfhydryl activity and structural stability of the metallothionein.

[0029] In this embodiment, the release efficiency of metallothionein in sturgeon liver is optimized by adding complex enzymes in stages and combining temperature and pH control. Neutral protease and flavor protease work synergistically, the former cuts the internal peptide bonds of the protein, and the latter degrades the hydrophobic terminal peptide chains, thereby more thoroughly hydrolyzing the liver tissue matrix and reducing the residual impurities that have not been degraded. The strategy of adding enzymes in stages can avoid self-consumption caused by excessive enzymes, maintain the activity in the late stage of enzymatic hydrolysis, and improve the yield of target protein. In addition, the gradient centrifugation and heat treatment steps after enzymatic hydrolysis can effectively separate impurities with poor thermal stability, and fix the conformation of metallothionein by rapid cooling, thereby reducing oxidative damage. The final freeze-drying process combined with low-temperature centrifugation can retain the activity of metallothionein to the greatest extent, providing high-purity raw materials for subsequent beverage formulations.

[0030] In another embodiment of the present invention, during the cutting process, the size of the sturgeon liver block can be set to 0.8-1.2 cm 3 The cube has a cutting tool edge angle of 25° and a feed speed of 20-25 pieces per minute. A food-grade stainless steel multi-head cutting machine can be used, equipped with a positioning mold with adjustable spacing, and the mold aperture matches the target size. Sturgeon liver comes from artificially farmed Acipenser schrenckii or Siberian sturgeon and is processed within 2 hours after slaughter to maintain freshness. The cutting machine is located in the raw material pretreatment area, connected to the conveyor belt, and directly connected to the crushing station downstream. The whole piece of sturgeon liver is fed into the cutting machine via the conveyor belt, guided by the positioning mold, and the double rows of rotating blades simultaneously complete the horizontal and vertical cutting, and the output block of liver tissue falls into the stainless steel hopper for temporary storage. In the staged crushing operation, the first crushing adopts the pulse mode, with a single working cycle of 5 seconds / rest of 3 seconds, a total duration of 1.8 minutes, and a blade speed of 3200 rpm; the second crushing adopts the continuous mode, lasts 2.5 minutes, and the blade speed is 4200 rpm. A high shear tissue crusher can be used, equipped with a 304 stainless steel blunt blade and a variable frequency speed motor, and a built-in temperature sensor to monitor the homogenate temperature in real time. The mass ratio of ice to water in the mixed medium of crushed ice and water is 3:1, the crushed ice particle size is ≤5mm, and the water temperature is initially set to 0-2℃. The crusher is installed on an independent seismic isolation platform, and the hopper and ice water tank are arranged side by side on both sides of the operating table. The pulse period and speed curve are preset by the PLC program, and the temperature sensor data is recorded every 10 seconds and triggers an over-temperature alarm (threshold ≥8℃). Three groups of liver block samples were taken, and the homogenate particle size distribution at different speeds was tested respectively, and the D 90 value. During the low-temperature resting step, the volume of the mixed medium is three times the volume of the liver block, and the resting time is 5-8 minutes, allowing the core temperature to drop to 5±0.5°C. A double-layer stainless steel ice water tank with a 50L inner tank, an outer layer filled with polyurethane insulation, and a bottom drain valve can be used. Crushed ice can be prepared on-site using an ice maker, and the water quality meets the GB 5749 standard for drinking water. The ice water tank is located to the side of the blender and connected to the blender hopper via a slide rail, facilitating rapid material transfer. The cut liver blocks slide through the hopper into the ice water tank and are immersed in the mixed medium. A temperature probe is inserted into the center of the liver block to monitor the cooling curve. Once the temperature reaches the specified value, a pneumatic push rod pushes the liver block to the blender inlet. This method uses staged mechanical fragmentation and precise temperature control to reduce frictional heat generation, maintain cell membrane integrity, and prevent metallothionein degradation caused by premature release of endogenous proteases.

[0031] In this embodiment, the refined processing of sturgeon liver tissue is achieved through staged mechanical crushing and temperature control. The first pulse crushing combined with intermittent cooling can avoid local overheating caused by continuous high-speed shearing and reduce the risk of protein thermal denaturation; the second continuous high-speed crushing further refines the tissue particles and improves the homogenate uniformity. The combination of block cutting and low-temperature stasis can not only inhibit the autolysis of endogenous enzymes, but also maintain the integrity of the cell structure, ensuring the targeted release of metallothioneins during the subsequent enzymatic hydrolysis process. This method improves the crushing efficiency while reducing the damage to the target protein caused by mechanical energy input, laying the foundation for efficient extraction.

[0032] In another embodiment of the present invention, ultrasonic treatment uses an ultrasonic generator with a frequency of 20-25kHz and a power density of 50-80W / L. A tank-type ultrasonic device can be used, and its probe is immersed 1-2 cm below the surface of the tissue homogenate. The treatment time is 8-12 minutes. The treatment tank is made of 316L stainless steel with a smooth inner wall to reduce energy loss. The ultrasonic generator can be installed on the side of the operating table, and the frequency and power can be adjusted through the control panel to display the treatment status in real time. During the treatment process, the temperature of the tissue homogenate is maintained at 4-6°C by an external cooling circulator. The cooling medium is an ethylene glycol aqueous solution, and the circulation pipeline is connected to the treatment tank interlayer. After ultrasonic treatment, the tissue homogenate is immediately transferred to a constant-temperature water bath with a built-in heating coil and temperature sensor. The temperature is raised at a controlled rate of 2-3°C per minute to a target temperature of 50-55°C. A stirring paddle located at the bottom of the water bath stirs at a low speed of 50-100 rpm to promote temperature uniformity. A temperature sensor probe is inserted into the center of the homogenate, and data is fed back to the temperature control system for precise regulation. The heating process should be completed within 5-8 minutes to avoid sudden temperature increases that may cause protein denaturation. The ultrasonic treatment tank is connected to the constant-temperature water bath via sealed piping lined with food-grade silicone to prevent cross-contamination. After ultrasonic treatment, the tissue homogenate is transferred to the water bath via a pneumatic diaphragm pump at a speed of 10-15 L / min to minimize transfer time. The water bath is preheated to 50°C for standby use, ensuring a seamless temperature ramp-up process. The cooling system of the ultrasonic treatment tank operates independently from the heating system to prevent energy interference. In this implementation, ultrasonic pretreatment accelerates cell disruption through physical cavitation, releasing more metallothionein precursors. Low temperatures minimize bacterial growth and protein degradation, ensuring raw material activity. Rapid temperature increases shorten process cycles and reduce energy consumption. This process optimizes enzymatic hydrolysis efficiency and provides a high-purity hydrolyzate for subsequent purification.

[0033] In another embodiment of the present invention, when adjusting the pH, the initial pH of the tissue homogenate can be pre-adjusted to 6.0±0.2 with 0.1M citric acid-disodium hydrogen phosphate buffer. The buffer solution is injected into the reaction vessel at a rate of 5-10mL / min by a peristaltic pump. The container is made of 316L stainless steel and has an anchor stirring paddle that mixes at a uniform speed of 60-80 rpm. Subsequently, a 0.1M sodium bicarbonate solution is added dropwise at a rate of 0.5mL / min using a microinjection pump, and the target pH value is 6.8±0.1. The pH meter probe is installed below the side wall of the container, 10-15 cm from the liquid surface, and the real-time data is linked to the dripping system through the controller to avoid excessive local acid and alkali concentrations.

[0034] The protective agent is a mixture of L-cysteine and reduced glutathione in a 3:1 molar ratio, dissolved in phosphate buffer at pH 6.5 to a concentration of 0.1-0.2 M. The dosage is 0.02%-0.05% of the mass of the tissue homogenate, and the enzymatic solution is added every 45 minutes via an automated dosing system. The dosing head is located in the center of the top of the reaction vessel, and the tubing is made of polytetrafluoroethylene to prevent metal ion contamination. After addition, the stirring speed is increased to 80-100 rpm and maintained for 3 minutes to ensure uniform dispersion. The protective agent can be made of food-grade L-cysteine and glutathione lyophilized powders and stored in a dark place.

[0035] The reaction vessel is equipped with a nitrogen blanketing system. The air inlet is located at the bottom of the vessel, and the nitrogen flow rate is controlled at 0.5-1.0L / min to maintain the headspace oxygen content below 5%. The agitator shaft seal uses a double-end mechanical seal to prevent air infiltration. When adding protective agent, the liquid addition line and the nitrogen line are opened simultaneously to form an inert gas protective layer. The temperature of the enzymatic hydrolyzate is maintained at 50±1°C by the interlayer circulating water. The circulating water inlet is located at the bottom of the vessel, and the outlet is located on the upper side wall to form a stable heat exchange. In this implementation, precise pH control minimizes enzyme activity loss and protein structural damage; periodic addition of a thiol-protecting agent effectively maintains the reduced-state activity of metallothionein; and nitrogen protection and a sealed design inhibit oxidative side reactions. This process enhances the stability and biological activity of the enzymatic hydrolysis product, providing a high-purity, low-oxidation metallothionein raw material for subsequent purification.

[0036] In another embodiment of the present invention, during the pre-activation treatment of the complex enzyme, the neutral protease and flavor protease can be activated for 15 minutes at 40°C ± 0.5°C and pH 7.0 ± 0.1, respectively. A constant temperature water bath equipped with an online pH monitoring module and a magnetic stirring device can be used, and the bath medium is deionized water. The neutral protease activity is ≥ 500,000 U / g, and the flavor protease activity is ≥ 200,000 U / g. The two are dry-mixed in a mass ratio of 1:1 and stored in a dry, light-proof container. The activation device is located in the pretreatment area of the enzymatic hydrolysis workshop and is connected to the enzymatic hydrolysis tank via sterile piping. During the activation process, the enzyme powder is mixed with the preheated buffer solution, and the magnetic stirring speed is set to 120 rpm. After activation is completed, the enzyme powder is transported to the enzymatic hydrolysis tank via a pneumatic diaphragm pump. In the staged stirring control, the stirring speed can be set to 160±5 rpm after the first addition of the complex enzyme, and adjusted to 190±5 rpm after the second enzyme addition. A variable frequency speed regulating stirring motor can be selected, and the drive shaft is connected to a double-layer inclined blade turbine blade, with the ratio of the blade diameter to the inner diameter of the enzymatic hydrolysis tank being 1:3. The stirring motor is installed at the flange on the top of the enzymatic hydrolysis tank, and the temperature control probe is located on the side wall of the tank at 1 / 3 of the height from the bottom. The two-stage stirring parameters are set by the PLC program, and the operation is 1.5 hours after the first enzyme addition and 1.5 hours after the second enzyme addition. During the experimental verification, three groups of enzymatic hydrolysis solution samples were taken, and the amount of metallothionein released at different stirring speeds was measured respectively, and the target protein concentration was detected by ELISA. During the temperature gradient adjustment process, the temperature is controlled at 51±0.3℃ for the first 2 hours, and then rises to 54.5±0.3℃ in the next 1.5 hours, with a heating rate of 2℃ per hour. A jacketed enzymatic hydrolysis tank can be used, and the external circulation heat transfer oil system is equipped with a PID temperature control module, and the heat transfer oil is food-grade silicone oil. The temperature sensor probe is inserted into the enzymatic hydrolysis solution at a depth of 10cm below the liquid surface, at an angle of 30° to the central axis of the tank. The oil inlet of the jacket of the enzymatic hydrolysis tank is located at the bottom, and the oil return port is located at the top, forming a countercurrent heat exchange. During the functional test, the uniformity of the temperature distribution in the tank is monitored by an infrared thermal imager, and the temperature difference is controlled within ±0.5℃. This process reduces the damage of high temperature to metallothionein by matching the temperature sensitive range of enzyme activity, while improving the thermal denaturation efficiency of impurity proteins.

[0037] In this implementation, the catalytic efficiency of the complex enzyme was optimized through enzyme preactivation and a staged stirring strategy. After activation, the neutral protease and flavor protease rapidly reached a highly active state, shortening the lag phase of the enzymatic hydrolysis reaction. Adjustment of the stirring speed in two stages matched the requirements of the enzymatic hydrolysis process: initial low-speed stirring prevented excessive shear damage to the released metallothioneins, while accelerated stirring in the later stages promoted further hydrolysis of undegraded tissue residues. The temperature gradient design (50-52°C in the early stages, 54-55°C in the later stages) balanced the optimal range of enzyme activity with the thermal stability of the target protein, minimizing the co-dissolution of contaminants.

[0038] In another embodiment of the present invention, the filtrate is initially centrifuged at 8,000-10,000 rpm for 5-8 minutes at 4°C using a high-speed refrigerated centrifuge with polypropylene tubes and a fixed-angle rotor. The centrifuge is installed in a low-temperature operating room (4±1°C). After centrifugation, the supernatant is transferred to a clean container via a siphon, and the sediment is discarded. A second centrifugation is performed at 10,000-12,000 rpm for 10-15 minutes using highly transparent polycarbonate tubes to facilitate observation of the liquid separation. The supernatant is aspirated using a sterile pipette to avoid disturbing the sediment.

[0039] After centrifugation, the supernatant is filtered through a 0.22 μm sterile filter membrane, preferably a polyethersulfone membrane, installed in a stainless steel filter housing. The filtration pressure is controlled at 0.1-0.3 MPa. The filter inlet and outlet pipes are connected to sanitary clamps, and the sealing ring is made of EPDM rubber. Before filtration, pre-rinse the filter membrane with sterile water for injection to remove bubbles and moisten the membrane surface. The final filtrate is collected in a pre-sterilized glass bottle, covered with sterile aluminum foil, and temporarily stored in a refrigerator at 4°C. The freeze-drying protective agent is a mixture of trehalose and mannitol in a mass ratio of 1:2 and dissolved in the final filtrate to a final concentration of 5%-8%. Trehalose can be used in food-grade crystals, and mannitol is injection-grade powder. During dissolution, a magnetic stirrer is used at 300-500 rpm for 20-30 minutes. The mixture is transferred to a freeze-drying tray made of medical-grade stainless steel with a thickness of 2-3 mm. The loading capacity is controlled to a liquid layer height of 1-1.5 cm. The solution after adding the protective agent is pre-frozen to -45°C in the freeze dryer and maintained for 4 hours before the main drying stage.

[0040] In this embodiment, multi-stage centrifugation and terminal filtration are combined to effectively remove residual impurities and microorganisms, ensuring the sterility of the final filtrate; the glassification effect of the lyophilization protectant reduces protein structural damage and improves the solubility and activity retention of the lyophilized product; the overall process meets pharmaceutical-grade production standards and is suitable for the long-term stable storage and transportation needs of metallothioneins.

[0041] In another embodiment of the present invention, the mixed fruit puree consists of blueberry puree (30-40% by volume), mulberry puree (25-35% by volume), and black wolfberry puree (the remainder). The puree can be a commercially available aseptically packaged product or homemade juice. For homemade juice, the blueberries, mulberries, and black wolfberries are washed and squeezed using a screw juicer at a pressure of 20-25 MPa. The pomace is then filtered through a 100-mesh filter to remove the residue. The puree is temporarily stored in a 4°C refrigerated tank made of food-grade stainless steel. The inlet and outlet are located at the bottom of the tank, and the flow is controlled by a sanitary butterfly valve.

[0042] The raw slurry is added to a mixing tank with a capacity of 500-1000L. The tank is equipped with three layers of pitched-blade turbine impellers, with a blade diameter to tank diameter ratio of 1:3. The stirring speed is set at 200-300 rpm for 15-20 minutes. Circulating water is passed through the tank interlayer to maintain a temperature of 10-15°C. The mixed slurry is pumped through a pipe into a homogenizer under two homogenization pressures: the first stage is 25-28 MPa with a homogenizer head gap of 0.1 mm; the second stage is 18-22 MPa with a gap of 0.25 mm. An online viscometer is installed at the homogenizer outlet to monitor slurry fluidity in real time. After homogenization, the mixed pulp is transferred to a temporary storage tank, where it is treated with an ultrasonic defoamer (28kHz frequency, 50W / L) for 5-8 minutes to eliminate bubbles and improve texture uniformity. The temporary storage tank is connected to a plate heat exchanger, where the pulp is cooled to 4-6°C before sampling and testing for pH (3.2-3.8) and soluble solids (12-15°Bx). Once qualified, the pulp is transported to the filling line via sterile piping, where the interior of the piping is electrolytically polished to a roughness Ra ≤ 0.4μm. In this embodiment, the specific proportion of the mixed fruit pulp is designed to balance the natural fruit aroma and the distribution of functional ingredients. The sourness and sweetness of blueberries and mulberries neutralize the slight bitterness of black wolfberries; the two-stage homogenization and ultrasonic treatment synergistically improve the beverage's taste and suspension stability; low-temperature mixing and aseptic transportation ensure the retention of the active ingredients of the raw materials, meeting the production requirements of ready-to-drink functional beverages.

[0043] In another embodiment of the present invention, blueberry puree, mulberry puree, and black wolfberry puree are added to a blending tank at a volume percentage of 30-40%, 25-35%, and the remainder. A stainless steel stirring tank with a capacity of 500-1000L can be used, and a three-layer inclined blade turbine stirring paddle is installed in the tank, and the ratio of the blade diameter to the tank diameter is 1:3. The stirring speed is set to 200-300 rpm, and the mixture is mixed for 15-20 minutes to form a homogeneous pulp. Metallothionein powder is then added to a final concentration of 95-105 mg / mL and stirring is continued for 10-15 minutes. Sucrose and citric acid are added at 3-5% and 0.1-0.3% of the total mass of the mixed pulp, respectively. Sucrose is food-grade white sugar, and citric acid is anhydrous crystalline particles. After dissolution, they are injected into the bottom of the blending tank through a pipeline.

[0044] The mixed liquid is pumped to a high-pressure homogenizer via a centrifugal pump. The homogenization pressure is divided into two stages: the first stage is 25-28 MPa, with a homogenization chamber gap of 0.1 mm; the second stage is 18-22 MPa, with a gap of 0.25 mm. The homogenizer outlet is connected to a plate heat exchanger, preheated to 75-80°C, and then enters a high-temperature instantaneous sterilization device. The sterilization conditions are 120-130°C for 3-5 seconds. After sterilization, the mixed liquid is cooled to 25-30°C within 45 seconds via a spiral thin-layer heat exchanger. The refrigerant in the heat exchanger is a 2-5°C ethylene glycol-water solution, with a flow rate matching of 1:1.2 (feed liquid to refrigerant volume ratio). The cooled beverage is transported to the filling line via sterile piping. The filling head is positioned 10-15 cm above the glass bottles, and the bottles are steam sterilized at 121°C for 20 minutes. The mixed liquid temperature is maintained at 25-30°C during filling, and the filling volume tolerance is controlled within ±1%. Before capping, nitrogen is injected into the bottle headspace through a nitrogen nozzle at a flow rate of 0.8-1.2 L / min, reducing the residual oxygen content in the headspace to below 0.8%. The capping machine torque is set to 8-12 N·m, and the sealing gasket is made of food-grade silicone. After pre-sterilization, the caps are conveyed in a directionally controlled manner via a vibrating plate. In this implementation, two-stage homogenization refines pulp particles and protein aggregates, improving the beverage's texture uniformity. High-temperature instantaneous sterilization inactivates microorganisms while reducing the loss of heat-sensitive ingredients. Nitrogen replacement inhibits oxidative deterioration and extends the product's shelf life. This process enables efficient and continuous production, meeting the industrial quality requirements of functional beverages.

[0045] In another embodiment of the present invention, vitamin C and vitamin E are pre-dry blended in a 2:1 mass ratio using a V-type blender for 20-30 minutes at a speed of 10-15 rpm. The blender hopper has a capacity of 50-100 L, a mirror-polished stainless steel inner wall, a mixing shaft located at the center axis of the hopper, and a 45° paddle angle. Vitamin C is food-grade L-ascorbic acid powder, and vitamin E is d-α-tocopherol oil, adsorbed onto a microcrystalline cellulose carrier to form a flowable powder. After dry blending, the mixture is passed through a vibrating sieve (80-100 mesh) to remove lumps and temporarily stored in a light-proof aluminum foil bag.

[0046] β-cyclodextrin is mixed with 40-45°C deionized water in a 1:10 weight-to-volume ratio. A stainless steel swelling tank with interlayer heating can be used. Initially, stir at 100-150 rpm. Raise the water bath temperature at 1°C / minute to 40°C and maintain this temperature for 15 minutes. Then, raise the temperature at 2°C / minute to 60°C, reduce the stirring speed to 80-100 rpm, and maintain this temperature for 10 minutes. The swelling tank should be equipped with an anchor-type stirring paddle, 5-8 cm from the bottom. An online turbidimeter should be installed in the light-transmitting window of the tank. Heating should be stopped when the transmittance reaches 90%. After cooling the solution to 45-50°C, filter it through a 100-mesh filter bag to obtain a transparent inclusion solution.

[0047] Add the vitamin mixture and β-cyclodextrin inclusion solution to the mixing tank at 0.05-0.1% and 0.8-1.2% of the total weight of the mixed fruit pulp, respectively. Also add the remaining sucrose and citric acid. The addition port is located on the upper side of the mixing tank, 15-20 cm from the liquid surface, and a fan-shaped nozzle is used for atomization. Maintain a stirring speed of 200-250 rpm. The tank temperature is controlled at 55-60°C via interlayer circulating water. Mix for 25 minutes. Maintain a linear velocity of 2.5-3.0 m / s at the end of the agitator to create a composite axial-radial flow pattern, ensuring uniform dispersion of the additives. In this embodiment, the antioxidant network effect is enhanced through the synergistic effect of vitamins C and E, and the oxidative degradation of functional ingredients is delayed; β-cyclodextrin inclusion improves the dispersion stability of fat-soluble components and reduces stratification during storage; the temperature-controlled mixing process ensures the activity of heat-sensitive ingredients and improves the comprehensive stability and functional performance of the beverage formula.

[0048] In another embodiment of the present invention, a stainless steel swelling tank with interlayer heating can be used for β-cyclodextrin pretreatment. The initial water temperature is set at 40-45°C, and the feed ratio is 1:10 (β-cyclodextrin mass to deionized water volume). The water bath is heated at a rate of 1°C / minute to 40°C. An anchor-type agitator is used within the tank to stir at a low speed of 100-150 rpm, with the blade 5-8 cm from the tank bottom. After maintaining the constant temperature for 15 minutes, the heating rate is adjusted to 2°C / minute to 60°C, and the stirring speed is reduced to 80-100 rpm for 10 minutes. A temperature sensor probe is located in the middle of the tank sidewall, and data is fed back to the temperature control system for precise regulation. An online turbidity meter is installed in the light-transmitting window of the swelling tank to monitor the solution's transmittance in real time. When the transmittance reaches 90%, heating and stirring are discontinued. The solution is filtered through a 100-mesh nylon filter bag installed at the tank's bottom outlet flange, with a filtration pressure not exceeding 0.1 MPa. After filtration, the solution is naturally cooled to 45-50°C and temporarily stored in an insulated storage tank. Recirculating water is passed through the tank's interlayer to maintain a temperature fluctuation of ±1°C. β-cyclodextrin should be food-grade, with a purity of ≥98% and a moisture content of ≤5%.

[0049] The transparent inclusion solution is pumped to the blending tank via a sanitary diaphragm pump at a controlled speed of 5-8 L / min. The inner surface of the pipe is electrolytically polished (roughness Ra ≤ 0.8 μm). During delivery, the solution temperature is maintained at 45-50°C via a heating sleeve using a hot water circulation system (set temperature 50 ± 2°C). The inlet of the blending tank is located 20 cm above the liquid surface, and an umbrella distributor is used to disperse the solution to avoid localized high concentrations. The pipeline length between the swelling tank and the blending tank does not exceed 3 meters to minimize heat loss during delivery. In this embodiment, gradient temperature increase is used to promote the full expansion of the β-cyclodextrin molecular cavity, thereby improving the inclusion efficiency of hydrophobic components; transmittance monitoring ensures complete swelling to prevent undissolved particles from affecting the clarity of the final product; low-temperature transportation and dispersion design ensures the stability of the inclusion solution, providing a uniform functional ingredient carrier for the beverage, while masking unpleasant flavors and optimizing taste coordination.

[0050] Example 1: The preparation method of metallothionein comprises the following steps: Cut fresh sturgeon liver into pieces (1cm 3 The liver was pre-cooled to 4°C and homogenized using a tissue grinder (3000 rpm for 4 minutes). The enzyme complex (neutral protease and flavor protease mixed in a 1:1 mass ratio, totaling 0.75% of the liver mass) was added in two batches: 60% of the enzyme was added initially, and the remaining 40% was added after 1 hour of enzymatic hydrolysis. Enzymatic hydrolysis was maintained at 50°C for 4 hours with a stirring speed of 150 rpm. The hydrolyzate was centrifuged at 4°C and 9000 rpm for 15 minutes. After centrifugation, the solution separated into three layers: an upper lipid layer, a clear enzymatic supernatant in the middle, and a lower layer containing precipitated solids. The supernatant was initially filtered through gauze to remove any remaining fine solid particles. The filtered supernatant was then filtered through a 0.45μm microporous membrane to obtain a relatively clear filtrate. The filtrate was poured into a beaker and heated in an 82°C water bath for 12 minutes to denature and precipitate most of the impurities. Stir continuously during heating to ensure uniform heating. After heating, the beaker was quickly cooled to room temperature in an ice bath. The mixture was then centrifuged at 11,000 rpm for 12 minutes at 4°C to remove precipitated proteins. The supernatant was collected and lyophilized directly. Protein concentration was determined using the BCA assay, and the metallothionein (MT) yield per kilogram of sturgeon liver was calculated. The antioxidant activity of metallothionein was assessed based on ABTS free radical scavenging rate, with the activity unit defined as the amount of protein required to scavenge 50% of free radicals (U / mg). The extraction yield of metallothionein was 28.6 mg / kg, with an activity of 155 U / mg.

[0051] Example 2: The preparation method of metallothionein comprises the following steps: Cut the sturgeon liver into pieces (0.5 cm 3 The fragments were crushed twice: the first time using a pulse mode (5 seconds on / 3 seconds off, 3000 rpm, for 1.5 minutes), immediately transferred to an ice-water mixture and cooled to 4°C; the second time using a continuous mode (4000 rpm, for 2 minutes). Subsequent enzymatic hydrolysis, centrifugation, and lyophilization steps were the same as in Example 1. The final metallothionein extraction yield was 31.2 mg / kg, with an activity of 170 U / mg.

[0052] Example 3: Based on Example 2, the tissue homogenate was ultrasonically pretreated for 10 minutes at 4°C using an ultrasonic device with a frequency of 25 kHz and a power density of 60 W / L. Subsequent enzymatic hydrolysis, centrifugation, and lyophilization procedures were the same as in Example 2. The final metallothionein extraction yield was 35.4 mg / kg, with an activity of 185 U / mg.

[0053] Example 4: Based on Example 3, the initial pH of the tissue homogenate was pre-adjusted to 6.0 using 0.1 M citric acid-disodium hydrogen phosphate buffer, and 0.1 M sodium bicarbonate solution was added dropwise at a rate of 0.5 mL / min via a microinjection pump to a pH of 6.8. During the enzymatic hydrolysis process, a mercaptoethanol protective agent (L-cysteine and reduced glutathione in a 3:1 molar ratio) was added at 0.03% by weight of the tissue homogenate. Subsequent steps were identical to those in Example 3. The final metallothionein extraction yield was 38.7 mg / kg, with an activity of 200 U / mg.

[0054] Example 5: Based on Example 4, neutral protease and flavor protease were activated separately at 40°C and pH 7.0 for 15 minutes and then mixed. Enzymatic hydrolysis was performed in two stages: after the initial enzyme addition, stirring was performed at 160 rpm (50°C for the first 2 hours); after the second enzyme addition, the temperature was raised to 54°C and the stirring speed was increased to 190 rpm. Subsequent steps were the same as in Example 4. The final metallothionein extraction yield was 41.5 mg / kg, with an activity of 215 U / mg.

[0055] Example 6: Based on Example 5, the filtrate was subjected to multiple centrifugation stages: first, centrifugation at 8000 rpm for 8 minutes at 4°C to discard the precipitate, and then the supernatant was centrifuged at 12000 rpm for 10 minutes. The supernatant after the second centrifugation was filtered through a 0.22 μm sterile filter membrane, and a lyoprotectant (trehalose and mannitol mixed in a 1:2 mass ratio, added at 6% of the protein weight) was added. The lyophilization procedure included a pre-freezing stage (maintaining at -40°C for 4 hours) and a main drying stage (gradually increasing the temperature to 20°C). The final metallothionein extraction yield was 45.2 mg / kg, with an activity of 235 U / mg.

[0056] Comparative Example 1: The preparation method of metallothionein comprises the following steps: Cut the sturgeon liver into pieces (1 cm 3The liver was pre-cooled to 4°C and crushed into a homogenate using a tissue grinder (3000 rpm for 4 minutes). The entire enzyme complex (neutral protease and flavor protease, mixed in a 1:1 mass ratio, totaling 0.75% of the liver mass) was added to the homogenate at once. Enzymatic digestion was carried out at 50°C for 4 hours with stirring at 150 rpm. The hydrolyzate was centrifuged at 4°C and 9000 rpm for 15 minutes. After centrifugation, the solution separated into three layers: an upper lipid layer, a clear enzymatic supernatant in the middle, and a lower layer containing precipitated solids. The supernatant was initially filtered through gauze to remove any remaining fine solid particles. The filtered supernatant was then filtered through a 0.45μm microporous membrane to obtain a relatively clear filtrate. The filtrate was poured into a beaker and heated in an 82°C water bath for 12 minutes to denature and precipitate most of the impurities. Stir continuously during heating to ensure uniform heating. After heating, the beaker was quickly cooled to room temperature in an ice bath. The mixture was then centrifuged at 11,000 rpm for 12 minutes at 4°C to remove precipitated proteins. The supernatant was collected and lyophilized. The final metallothionein extraction yield was 23.5 mg / kg, with an activity of 128 U / mg.

[0057] As can be seen, compared with Comparative Example 1, Example 6, through optimization steps such as staged enzyme addition, ultrasonic pretreatment, precise pH control, enzyme preactivation, multi-stage centrifugation, and lyophilization protectant, increased the metallothionein extraction rate from 23.5 mg / kg to 45.2 mg / kg (a 92.3% increase), and increased the metallothionein activity from 128 U / mg to 235 U / mg (an 83.6% increase). This demonstrates that the multi-technique synergy of the present invention significantly overcomes existing process bottlenecks, achieving the advantages of both efficient extraction and high activity retention.

[0058] Example 7: An anti-ultraviolet radiation beverage comprises a mixed fruit pulp and metallothionein at a concentration of 100 mg / mL. The metallothionein is prepared using the method of Example 6. The mixed fruit pulp comprises, by volume, 35% blueberry puree, 30% mulberry puree, and 35% black wolfberry puree. Preparation of blueberry puree: Select fresh, ripe, pest-free blueberry fruits, wash them with clean water, and remove the stems and impurities. The blueberry fruits are crushed in a crusher and then squeezed through a juicer to obtain blueberry juice. The blueberry juice is then filtered to remove pulp residue and seeds, obtaining clarified blueberry puree. The blueberry puree is pasteurized at 68°C for 18 minutes to kill microorganisms and extend its shelf life. Preparation of mulberry puree: Select mature, high-quality mulberry fruits, rinse them with running water, and drain. The mulberries are blended in a pulper and further refined using a colloid mill to achieve finer pulp particles. The pulp is then filtered through a mesh to remove larger particles, resulting in mulberry puree. The mulberry puree is also pasteurized at 68°C for 18 minutes. To prepare black wolfberry puree, high-quality black wolfberries are soaked in warm water for 12 minutes to remove surface dust and impurities. Remove the soaked black wolfberries, drain, and extract the juice in a juicer. After extraction, the black wolfberry juice is centrifuged at 3500 rpm for 8 minutes to remove insoluble impurities, resulting in black wolfberry puree. This black wolfberry puree is also pasteurized at the same temperature and time as the blueberry and mulberry purees.

[0059] The preparation method of the anti-ultraviolet radiation beverage is: (1) Allocation Add the prepared blueberry puree, mulberry puree, and black wolfberry puree to a mixing tank according to the aforementioned volume percentages (35% blueberry puree, 30% mulberry puree, and 35% black wolfberry puree). Stir at 250 rpm for 18 minutes to form a mixed fruit puree. Then, add metallothionein powder to the mixing tank to a final concentration of 100 mg / mL. Stir for 12 minutes to thoroughly mix the metallothionein with the mixed fruit puree. Next, add appropriate amounts of sweetener and acidulant, such as 4% (mass percentage) sucrose and 0.2% (mass percentage) citric acid, depending on the desired flavor. Stir at 180 rpm at 55°C for 25 minutes to obtain the prepared beverage.

[0060] (2) Homogenization The prepared beverage is transported to a high-pressure homogenizer through a pipeline and subjected to two-stage homogenization treatment at 25MPa and 45°C. The first-stage homogenization pressure is 26MPa and the homogenization chamber gap is 0.1mm. The second-stage homogenization pressure is 20MPa and the homogenization chamber gap is 0.25mm. The interval between the two-stage homogenization treatments does not exceed 5 minutes.

[0061] (3) Sterilization and filling The homogenized beverage is preheated to 78°C through a plate heat exchanger and then piped to a sterilizer for high-temperature instantaneous sterilization at 125°C for 4 seconds to kill harmful microorganisms in the beverage. The sterilized beverage is immediately cooled to 25-30°C within 45 seconds through a spiral thin-layer heat exchanger; the cooled mixture is filled into glass bottles that have been steam sterilized at 121°C for 20 minutes, maintaining the temperature of the mixture at 25-30°C during filling. Immediately after filling, the headspace is replaced with nitrogen to a residual oxygen content of ≤0.8%, and the bottles are sealed with screw caps to prevent microbial contamination.

[0062] Example 8: On the basis of Example 7, in the step of adding sucrose and citric acid, 0.08% of vitamin C, 0.04% of vitamin E and 1.0% of untreated β-cyclodextrin were added to the total mass of the mixed pulp. When adding vitamin C and vitamin E, the two were pre-dry mixed and then added.

[0063] Example 9: Based on Example 8, β-cyclodextrin was pretreated before being added, specifically comprising the following steps: β-cyclodextrin was mixed with 40°C water in a mass-to-volume ratio of 1:10, stirred and swelled in a water bath at 120 rpm, the water temperature was controlled to rise from the initial temperature to 40°C at a rate of 1°C per minute, and maintained at this temperature for 15 minutes; then the temperature was raised to 60°C at a rate of 2°C per minute, the stirring speed was adjusted to 90 rpm, and the stirring was maintained at a constant temperature for 10 minutes; heating was stopped when the transmittance of the solution reached more than 90%, and the solution was naturally cooled to 45°C under continuous stirring to form a transparent inclusion solution and immediately transferred into a mixing tank.

[0064] Comparative Example 2: An anti-ultraviolet radiation beverage, which is different from Example 7 in that the metallothionein is prepared by the method of Comparative Example 1.

[0065] Comparative Example 3: An anti-ultraviolet radiation beverage, which is different from Example 7 in that the beverage does not contain metallothionein powder.

[0066] Comparative Example 4: An anti-ultraviolet radiation beverage, which is different from Example 7 in that the mixed fruit pulp is replaced by water, that is, the beverage does not contain mixed fruit pulp.

[0067] Experimental Example 1: The beverages prepared in Examples 7-9 and Comparative Examples 2-4 were tested and verified for their anti-ultraviolet effects.

[0068] 1. Detection model selection This experiment used Caenorhabditis elegans (C. elegans) as a model organism to examine the effects of UV radiation. C. elegans has advantages such as ease of cultivation, a short life cycle, a well-defined genetic background, and sensitivity to environmental changes. Its damage to UV radiation can be assessed using behavioral indicators such as head shaking frequency and body flexion frequency.

[0069] (2) Testing process Nematode culture: C. elegans were plated onto NGM (Nematode Growth Medium) plates containing Escherichia coli OP50 as a food source and cultured in a 20°C incubator. The growth of the nematodes was regularly monitored. When nematodes reached an appropriate development stage (e.g., L4 larvae), they were used for subsequent experiments.

[0070] Grouping and Treatment: Cultured Caenorhabditis elegans were randomly divided into a normal group, a model group (UV), groups of Examples 7-9, groups of Comparative Examples 2-4, and a control group. The normal group was not exposed to UV light and did not receive the beverage of the present invention. The model group (UV) was exposed to UV light (254 nm, 590 μW / cm², 10 minutes) but did not receive the beverage. After UV light exposure, the nematodes in groups of Examples 7-9 and Comparative Examples 2-4 were exposed to the corresponding beverage for 48 hours. After UV light exposure, the nematodes in the control group were exposed to a standard UV-resistant beverage (containing 0.5% green tea polyphenols and no metallothionein) for 48 hours. Behavioral indicators of the nematodes were measured five days after the end of the treatment period.

[0071] Index detection Behavioral index detection: Each group selected 10 nematodes to be observed on a sterile NGM culture plate, and the experiment was carried out in parallel for 3 times. After the nematodes crawled for 1 minute to remove the impurities adhering to the body, the frequency of the nematodes' head shaking per minute was observed and recorded under a microscope. The nematodes' head swung from one side to the other and then returned to the original position, which was counted as one head shaking. Each group selected 10 nematodes to be observed on a sterile NGM culture plate, and the experiment was carried out in parallel for 3 times. After the nematodes crawled for 1 minute to remove impurities adhering to their bodies, the number of body bends per minute was observed and recorded under a microscope. The number of times the nematodes' heads returned to the direction in which they started crawling and the trunk completed a semicircular movement trajectory was counted as one body bend.

[0072] (3) Test results and analysis The test results are shown in the following table.

[0073] As shown in the table above, the head swing frequency in Example 7 (16.4 times / minute) was significantly higher than that in Comparative Example 3 (9.8 times / minute, without metallothionein), demonstrating that metallothionein is the core active ingredient in UV damage protection. Comparative Example 2 (single enzyme addition to prepare metallothionein) achieved a lower performance (12.6 times / minute) than Example 7 (16.4 times / minute), indicating that the staged enzyme addition process significantly enhances protein activity. The head swing frequency in Example 7 (16.4 times / minute) was 25% higher than that in Comparative Example 4 (13.1 times / minute, without fruit pulp), indicating that the antioxidant components (anthocyanins and polyphenols) in blueberries, mulberries, and black wolfberries synergistically scavenge free radicals with metallothionein. The head swing frequency in Example 8 (17.8 times / minute) was 8.5% higher than that in Example 7 (16.4 times / minute), indicating that the antioxidant network of vitamins C / E and β-cyclodextrin inclusion complexes reduce metallothionein inactivation. Example 9 (18.9 beats / minute) achieved a 6.2% improvement over Example 8 (unpretreated β-cyclodextrin), demonstrating that gradient temperature pretreatment enhances inclusion efficiency and protects the conformational stability of metallothionein. The head-shaking frequency (18.9 beats / minute) and body-bending frequency (9.7 beats / minute) in Example 9 approached those of the normal control group (19.5 and 10.8, respectively), and were 72.6% and 140% higher than those of the control group (11.3 and 4.5, respectively), significantly outperforming existing technologies. In summary, the present invention achieves UV protection close to normal physiological levels through the synergistic effects of metallothionein and mixed fruit pulp, the stabilization effect of vitamins / β-cyclodextrin, and the β-cyclodextrin pretreatment process. The nematode behavioral index recovery rate in Example 9 far exceeded that of the control and comparative examples, demonstrating that the present beverage can effectively mitigate UV damage to C. elegans and exhibits excellent UV protection.

[0074] The number of equipment and processing scales described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the UV-resistant beverage of the present invention will be apparent to those skilled in the art.

[0075] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. An anti-ultraviolet radiation beverage, characterized in that: include: The concentration is 95-105 mg / mL metallothionein, wherein the preparation method of the metallothionein comprises the following steps: Wash the fresh sturgeon liver, cut it into pieces, and crush it with a tissue crusher to form a tissue homogenate; The tissue homogenate is heated to 50-55° C., the pH value of the tissue homogenate is adjusted to 6.5-7.0, and a complex enzyme is added in two portions, with the first addition amount being 60%-70% of the total weight of the complex enzyme. After 1-1.5 hours of enzymatic hydrolysis, the remaining complex enzyme is added, wherein the complex enzyme comprises a neutral protease and a flavor protease in a mass ratio of 1:1, and the addition amount of the complex enzyme is 0.5%-1.0% of the weight of the sturgeon liver. After adding the complex enzyme, the enzymatic hydrolysis is carried out by stirring at a speed of 150-200 rpm for a total enzymatic hydrolysis time of 3-4 hours to obtain an enzymatic hydrolyzate. The enzymatic hydrolyzate was centrifuged at 8000-10000 rpm for 15-20 minutes at 4°C to obtain a supernatant; The supernatant was preliminarily filtered through gauze, and then filtered through a 0.45 μm microporous filter membrane to obtain a filtrate; The filtrate was heated in a water bath at 80-85°C for 10-15 minutes with continuous stirring, and then cooled to room temperature in an ice bath; The cooled filtrate is centrifuged at 4° C. and 10,000-12,000 rpm for 10-15 minutes, and the supernatant is collected and freeze-dried to obtain the metallothionein.

2. The anti-ultraviolet radiation beverage according to claim 1, characterized in that: Wash the fresh sturgeon liver and cut it into 0.5-1cm pieces 3 The liver was pounded into cubes in two steps using a tissue pounder. The first pounding time was 1.5-2 minutes in an intermittent pulse mode of 5 seconds on / 3 seconds off, with a pounder speed of 3000-3500 rpm. The second pounding time was 1.5-3 minutes in a continuous rotation mode, with a pounder speed of 4000-4500 rpm. Before each pounding, the sturgeon liver blocks or tissue homogenate were transferred to a mixture of crushed ice and water and allowed to stand until the core temperature dropped to 4-6°C.

3. The anti-ultraviolet radiation beverage according to claim 1, characterized in that: Before heating the tissue homogenate, the tissue homogenate is subjected to ultrasonic pretreatment, wherein the conditions of the ultrasonic pretreatment are: frequency 20-25 kHz, power density 50-80 W / L, and treatment time 8-12 minutes; wherein, during the ultrasonic pretreatment, the temperature of the tissue homogenate is maintained at 4-6°C.

4. The anti-ultraviolet radiation beverage according to claim 1, characterized in that: When adjusting the pH value of the tissue homogenate, first pre-adjust the pH to 6.0±0.2 with 0.1M citric acid-disodium hydrogen phosphate buffer, and then add 0.1M sodium bicarbonate solution dropwise at a rate of 0.5mL / min through a microinjection pump to the target pH value of 6.8±0.1; and after the first addition of the complex enzyme, add 0.02%-0.05% of the mass of the tissue homogenate to the enzymatic hydrolysis solution every 45 minutes. The mercaptoethanol protective agent is prepared by compounding L-cysteine and reduced glutathione in a molar ratio of 3:

1.

5. The anti-ultraviolet radiation beverage according to claim 1, characterized in that: The complex enzyme needs to be pre-activated before addition: neutral protease and flavor protease are activated separately at 40°C and pH 7.0 for 15 minutes, and then mixed in a ratio of 1:1; The stirring enzymolysis is divided into two stages: stirring at 150-170 rpm after the first addition of the complex enzyme, and stirring at 180-200 rpm after the second addition of the complex enzyme; the temperature is maintained at 50-52°C for the first 2 hours of the total enzymolysis time, and then raised to 54-5°C for the next 1-2 hours.

6. The anti-ultraviolet radiation beverage according to claim 1, characterized in that: Before centrifuging the cooled filtrate at 10,000-12,000 rpm at 4°C for 10-15 minutes, the filtrate is first centrifuged at 8,000-10,000 rpm at 4°C for 5-8 minutes, and after discarding the precipitate, the resulting supernatant is centrifuged at 10,000-12,000 rpm. After centrifugation, the supernatant is filtered through a 0.22 μm sterile filter membrane for terminal sterilization to obtain a final filtrate, and a lyoprotectant is added to the final filtrate before lyophilization. The lyoprotectant consists of trehalose and mannitol in a mass ratio of 1:2, and the added amount is 5%-8% of the protein amount in the final filtrate.

7. The anti-ultraviolet radiation beverage according to claim 1, characterized in that: Also includes: Mixed fruit pulp, wherein the mixed fruit pulp comprises, by volume percentage, 30-40% of blueberry puree, 25-35% of mulberry puree, and the remainder is black wolfberry puree.

8. The method for preparing the anti-ultraviolet radiation beverage according to claim 7, wherein: The following steps are involved: Add blueberry puree, mulberry puree, and black wolfberry puree according to the formulated amount into a mixing tank and stir at 200-300 rpm for 15-20 minutes to form a mixed fruit puree; add metallothionein powder to the mixed fruit puree to a final concentration of 95-105 mg / mL of metallothionein and continue stirring for 10-15 minutes; then add 3-5% sucrose and 0.1-0.3% citric acid of the total weight of the mixed fruit puree in sequence, and stir at 150-200 rpm at 50-60° C. for 20-30 minutes; The prepared mixed liquid is transported to a high-pressure homogenizer through a pipeline and subjected to two-stage homogenization treatment at a pressure of 20-30 MPa and a temperature of 40-50°C. The first-stage homogenization pressure is 25-28 MPa and the gap of the homogenization cavity is 0.1 mm. The second-stage homogenization pressure is 18-22 MPa and the gap of the homogenization cavity is 0.25 mm. The interval between the two-stage homogenization treatments does not exceed 5 minutes. The homogenized mixed liquid is preheated to 75-80°C through a plate heat exchanger, transported to a high-temperature instantaneous sterilization device through a centrifugal pump, maintained at 120-130°C for 3-5 seconds, and immediately cooled to 25-30°C within 45 seconds through a spiral thin-layer heat exchanger; the cooled mixed liquid is filled into glass bottles sterilized with 121°C steam for 20 minutes, and the temperature of the mixed liquid is maintained at 25-30°C during filling. Immediately after filling, the headspace air is replaced with nitrogen until the residual oxygen content is ≤0.8%, and the bottles are sealed with screw caps.

9. The method for preparing the anti-ultraviolet radiation beverage according to claim 8, wherein: It also includes, in the step of adding sucrose and citric acid, simultaneously adding 0.05-0.1% of vitamin C, 0.02-0.05% of vitamin E and 0.8-1.2% of beta-cyclodextrin based on the total mass of the mixed fruit pulp, wherein the vitamin C and vitamin E are pre-dry-mixed in a mass ratio of 2:1 and then added, and stirring is maintained at 55-60°C for 25 minutes.

10. The method for preparing the anti-ultraviolet radiation beverage according to claim 9, wherein: The β-cyclodextrin is pretreated before being added, specifically comprising the following steps: mixing the β-cyclodextrin with water at 40-45°C in a mass-to-volume ratio of 1:10, stirring and swelling the mixture in a water bath at 100-150 rpm, controlling the water temperature to rise from the initial temperature to 40°C at a rate of 1°C per minute, and maintaining this temperature for 15 minutes; subsequently heating the mixture to 60°C at a rate of 2°C per minute, adjusting the stirring speed to 80-100 rpm, and maintaining constant temperature stirring for 10 minutes; stopping heating when the solution transmittance reaches above 90%, and naturally cooling the mixture to 45-50°C under continuous stirring to form a transparent inclusion solution, and immediately transferring the solution into a mixing tank.

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